cAMP/CREB-regulated LINC00473 marks LKB1-inactivated lung cancer and mediates tumor growth
Zirong Chen, Jian-Liang Li, Shuibin Lin, Chunxia Cao, Nicholas T. Gimbrone, Rongqiang Yang, Dongtao A. Fu, Miranda B. Carper, Eric B. Haura, Matthew B. Schabath, Jianrong Lu, Antonio L. Amelio, W. Douglas Cress, Frederic J. Kaye, Lizi Wu
Zirong Chen, Jian-Liang Li, Shuibin Lin, Chunxia Cao, Nicholas T. Gimbrone, Rongqiang Yang, Dongtao A. Fu, Miranda B. Carper, Eric B. Haura, Matthew B. Schabath, Jianrong Lu, Antonio L. Amelio, W. Douglas Cress, Frederic J. Kaye, Lizi Wu
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Research Article Oncology

cAMP/CREB-regulated LINC00473 marks LKB1-inactivated lung cancer and mediates tumor growth

Abstract

The LKB1 tumor suppressor gene is frequently mutated and inactivated in non–small cell lung cancer (NSCLC). Loss of LKB1 promotes cancer progression and influences therapeutic responses in preclinical studies; however, specific targeted therapies for lung cancer with LKB1 inactivation are currently unavailable. Here, we have identified a long noncoding RNA (lncRNA) signature that is associated with the loss of LKB1 function. We discovered that LINC00473 is consistently the most highly induced gene in LKB1-inactivated human primary NSCLC samples and derived cell lines. Elevated LINC00473 expression correlated with poor prognosis, and sustained LINC00473 expression was required for the growth and survival of LKB1-inactivated NSCLC cells. Mechanistically, LINC00473 was induced by LKB1 inactivation and subsequent cyclic AMP–responsive element–binding protein (CREB)/CREB-regulated transcription coactivator (CRTC) activation. We determined that LINC00473 is a nuclear lncRNA and interacts with NONO, a component of the cAMP signaling pathway, thereby facilitating CRTC/CREB-mediated transcription. Collectively, our study demonstrates that LINC00473 expression potentially serves as a robust biomarker for tumor LKB1 functional status that can be integrated into clinical trials for patient selection and treatment evaluation, and implicates LINC00473 as a therapeutic target for LKB1-inactivated NSCLC.

Authors

Zirong Chen, Jian-Liang Li, Shuibin Lin, Chunxia Cao, Nicholas T. Gimbrone, Rongqiang Yang, Dongtao A. Fu, Miranda B. Carper, Eric B. Haura, Matthew B. Schabath, Jianrong Lu, Antonio L. Amelio, W. Douglas Cress, Frederic J. Kaye, Lizi Wu

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Figure 3

LINC00473 expression is regulated by the LKB1/CRTC1/CREB signaling axis.

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LINC00473 expression is regulated by the LKB1/CRTC1/CREB signaling axis....
(A) qRT-PCR analysis showed that LINC00473 expression was significantly reduced in two LKB1-null NSCLC cell lines (H157 and A549) after transduction with LKB1 retroviruses (LKB1) for 96 hours, with cells transduced with vector retroviruses (Ctl) used as controls. Western blotting confirmed LKB1 expression (n = 3, **P < 0.001). (B) LINC00473 expression was enhanced upon LKB1 shRNA lentiviral infection in two LKB1-WT NSCLC cell lines (H3123 and H322) (n = 3, *P < 0.05 and ***P < 0.0001). (C) LINC00473 expression was significantly reduced in A549 cells after transduction with two independent CREB shRNAs (n = 3, *P < 0.05). (D) A schematic representation of the LINC00473 promoter reporter. (E) Expression of LKB1, but not the kinase-dead K78I mutant in LKB1-null A549 cells caused significant repression of LINC00473 promoter reporter activity (n = 3, *P < 0.05). (F) Expression of A-CREB in A549 cells significantly inhibited the LINC00473 promoter (Pro) activity (n = 3, *P < 0.05). (G) Expression of WT or constitutively activated S151A CRTC1 increased LINC00473 promoter activity in LKB1-expressing H322 cells (n = 3, **P < 0.001 and ***P < 0.0001). (H) ChIP analysis indicated that CREB and CRTC1 were significantly enriched on the LINC00473 promoter encompassing the CRE half sites in A549 cells (n = 3, *P < 0.05 and ***P < 0.0001). See also Supplemental Figure 9.